Plasmodium vivax -infected blood samples. Blood samples obtained from patients with P. vivax infections at local hospitals in malaria-endemic areas of Korea (n = 97) during [2007][2008][2009]. Samples from Thailand (n = 30) were obtained from malaria patients who were admitted to a local health center in Mae Sod, Thailand, during 2008, and Abstract. Parasite dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) are known target enzymes of antifolate drugs used for the treatment and prophylaxis of persons with malaria. We sequenced the Plasmodium vivax dihydrofolate reductase ( pvdhfr ) and dihydropteroate synthase ( pvdhps ) genes to examine the prevalence and extent of point mutations in isolates from malaria-endemic countries. Double mutations (S58R and S117N) or quadruple mutations (F57L/I, S58R, T61M, and S117T) in the pvdhfr gene were found in isolates from Thailand (96.4%) and Myanmar (71.4%), but in only one isolate (1.0%) from Korea, where sulfadoxine-pyrimethamine has never been used. The pvdhfr point mutations correlated strongly with the pvdhps point mutations and ranged from single to triple mutations (S382A, A383G, and A553G), among isolates from Thailand, Myanmar, and Korea. These findings suggests that the prevalence of mutations in pvdhfr and pvdhps in P. vivax isolates from different malaria-endemic countries is associated with selection pressure imposed by sulfadoxine-pyrimethamine. DNA isolation. Genomic DNA was extracted from the whole blood samples by using a QIAamp DNA Blood Kit (Qiagen, Valencia, CA), according to the manufacturer's instructions and from blood filter papers by using described methods.
17Sequencing of pvdhfr and pvdhps genes . The pvdhfr (GenBank accession no. X98123) and pvdhps (AY186730) gene sequences used as the reference wild type were amplified by polymerase chain reaction (PCR) using gene-specific primers. Amplifications were performed in a reaction mixture that contained 2 μL of 10 × buffer, 2.5 mM MgCl 2 , 0.2 mM of each dNTP, 0.25 μM of each primer, 0.5 U of AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA), and 1 μL of genomic DNA or the amplicon from the first PCR. Primer Pvdhfr F1 (sense) 5′-ATGGAGGACCTTTCAGATGTATT-3′ and primer Pvdhfr R1 (antisense) 5′-CCACCTTGCTGTA AACCAAAAAGTCCAGAG-3′ (expected amplicon size = 715 basepairs, nucleotides 1-715) were used as primers for the first-round amplification of the pvdhfr gene. 18 The PCR was performed with an initial denaturation at 94°C for 10 minutes and 35 cycles at 94°C for 50 seconds, 58°C for 50 seconds, and 72°C for 50 seconds.Primer Pvdhfr F3 (sense) 5′-TTTGACATTTACGCCATC TGC-3′ and primer Pvdhfr R3 (antisense) 5′-TACACCTC ACTGACGGACG-3′ (expected amplicon size = 647 basepairs, nucleotides 22-668) were used for the second-round amplification of pvdhfr . The PCR cycling conditions for pvdhfr were an initial denaturation at 94°C for 10 minutes and 35 cycles at 94°C for 30 seconds, 60°C for 50 seconds, and 72°C for 1 minute.Primer Pvdhps F1 (sense) 5′-AGGAAGCCATTCGCTC AAC-3′ and pr...